Chemoenzymatic synthesis of trehalose analogues

ABSTRACT

The present invention provides methods of synthesizing trehalose analogues; methods of detecting mycobacteria, and trehalose analogues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/845,555, filed Dec. 18, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/641,007, filed Mar. 6, 2015, which claimspriority to U.S. Provisional Patent Application No. 61/949,688, filedMar. 7, 2014, the contents of which are hereby incorporated herein byreference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant number R15AI117670 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Trehalose is a C₂-symmetric disaccharide consisting of two glucosemolecules linked together by a 1,1-α,α-glycosidic bond. Althoughtrehalose is not present in mammals, it is widespread elsewhere innature, where it primarily functions as an energy source and as aprotectant against desiccation, osmotic stress, and changes intemperature. Trehalose metabolism is required for virulence in a numberof pathogenic organisms, most notably Mycobacterium tuberculosis (Mtb),which is the causative agent of human tuberculosis (TB). Mtb ischaracterized by its complex cell envelope, which harbors a variety oftrehalose glycolipids that are involved in cell-wall biosynthesis andthat contribute to pathogenesis. The essentiality of trehalosemetabolism in Mtb coupled with its absence in humans-makes it anattractive target for drug and diagnostic development, a notion that isunderscored by the recent identification of numerous antimycobacterialcompounds that inhibit trehalose glycolipid transport.

Despite their potential value, the development and application oftrehalose analogues in TB research remains limited, in large part due tothe difficulties associated with their chemical synthesis. Specifically,the C₂-symmetry and 1,1-α,α-glycosidic bond of trehalose posesignificant challenges. Methods for the desymmetrization andregioselective hydroxyl group manipulation of trehalose are usuallylengthy and low-yielding. On the other hand, methods for the formationof 1,1-α,α-glycosidic linkages are either laborious or suffer from lowstereoselectivity. In addition to these technical obstacles, multi-stepchemical synthesis of carbohydrates is often inefficient andinaccessible to non-experts.

A chemoenzymatic method for the synthesis of trehalose analogues wouldcomplement chemical methods and help to alleviate many of theseproblems. In general, enzymes can perform reactions with excellentregio- and stereoselectivity under mild conditions, and without the needfor protection of substrate functional groups. Moreover, enzymaticreactions are easy to carry out, non-hazardous, and environmentallybenign, making chemoenzymatic synthesis a cornerstone of “green”chemistry development. These attributes, coupled with the increasingfocus on trehalose and its derivatives in various scientific fields,motivated us to develop a robust chemoenzymatic approach to trehaloseanalogue synthesis.

SUMMARY

In one embodiment, the invention provides a method for synthesizingtrehalose analogues comprising contacting a glucose analogue with atrehalose synthase, a magnesium salt and a monosaccharide donor, withthe provisio that either the glucose analogue is not glucose or themonosachharide donor is not a glucose donor.

In one embodiment, the invention provides a method of detecting livemycobacteria in a sample comprising contacting a glucose analogue with atrehalose synthase, a magnesium salt and a monosaccaride donor to form atrehalose analogue; contacting a sample with the trehalose analogue;detecting the labeled mycobacteria; wherein the trehalose analogue islabeled with a detectable moiety.

In one embodiment, the invention provides a method of detectingmycobacteria in a cell comprising contacting a cell with a trehaloseanalogue; contacting a sample with the trehalose analogue; detecting thelabeled mycobacteria; wherein the trehalose analogue is labeled with adetectable moiety; and wherein the trehalose analogue accumulates in themycobacteria via the trehalose transport protein.

In one embodiment, the invention provides a compound according toformula (III) or (IV):

-   -   wherein    -   X is O, S, Se, PH, P(O)H, or P(O)OH;        -   R¹, R² and R³ are independently selected from H, halo, —OR,            —NR₂, —NR₃ ⁺, —NHCOR, —SR, —SCOR, —OSO₃ ⁻, —OPO₃ ²⁻, —SeR,            —SeCOR, —N₃, —CN, —NC, C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆            alkynyl;        -   R⁴ is OH, —NHR, —SH, or —SeH; and        -   each R is independently selected from H, C₁₋₆ alkyl, C₂₋₆            alkenyl or C₂₋₆ alkynyl;    -   with the proviso that the compound is not trehalose.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D show the synthesis of trehaloseanalogues from Glc analogues and UDP-Glc by TreT from T. tenax. In FIG.1A, FIG. 1B, FIG. 1C, and FIG. 1D yields were determined by HPLC andN.D. indicates not detected.

FIG. 2A and FIG. 2B. FIG. 2A shows the experimental workflow forsingle-day probe synthesis, metabolic labeling, and imaging of M.smegmatis, and in FIG. 2B fluorescence microscopy analysis of6-TreAz-treated (or untreated) M. smegmatis wild type, ΔsugC mutant, andΔsugC::sugC complement, indicating that trehalose analogue probes canaccumulate in mycobacteria via the trehalose-specific transporterSugABC-LpqY. TL, transmitted light. Scale bars, 5 m.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, and FIG. 3E. FIG. 3A shows theexperimental workflow for assessing uptake of fluoro-trehalose analoguesby M. smegmatis and in FIG. 3B, FIG. 3C, FIG. 3D, and FIG. 3E gaschromatograms for analysis of fluoro-trehalose analogue uptake in M.smegmatis wild type, ΔsugC mutant, and ΔsugC::sugC complement,indicating that trehalose analogue probes can accumulate in mycobacteriavia the trehalose-specific transporter SugABC-LpqY.

FIG. 4 shows the one-pot metabolic labeling.

DETAILED DESCRIPTION

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of construction and the arrangement of components set forthin the following description or illustrated in the following drawings.The invention is capable of other embodiments and of being practiced orof being carried out in various ways.

The present invention provides chemoenzymatic methods for synthesizingtrehalose analogues using a trehalose synthase (TreT) enzyme. In nature,TreT converts glucose and UDP-glucose into trehalose in a single step.The present invention uses analogues of glucose and/or UDP-glucose inthe TreT reaction to generate trehalose analogues. It is expected thatthe methods of the present invention will be useful for rapidly andefficiently preparing trehalose analogues as potentialanti-mycobacterial compounds, compounds for imaging/detection ofmycobacteria, and non-degradable biopreservation/bioprotectioncompounds, among other applications in the biological and materialssciences. For example, these methods could be used for transformingeasily accessible radiolabeled glucose analogues, e.g. [¹⁴C]-labeledglucose or [¹⁸F]-labeled glucose, into the corresponding [¹⁴C]- or[¹⁸F]-labeled trehalose analogues for investigating or imagingmycobacteria and other trehalose-containing organisms. These types oftrehalose analogues are virtually inaccessible using current synthesismethods or are exceedingly expensive to purchase. The methods of thepresent invention will provide a simple, fast (1 hour), andhigh-yielding (up to 99% yield) route to synthesizing trehaloseanalogues for various applications.

Definitions

As used herein, the term “alkyl” refers to a branched, unbranched, orcyclic hydrocarbon having, for example, from 1 to 6 carbon atoms or from1 to 4 carbon atoms. Examples include, but are not limited to, methyl,ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl,2-methyl-2-propyl (t-butyl), 1-pentyl, 2-pentyl, 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like. The alkyl canbe unsubstituted or substituted. The alkyl can also be optionallypartially or fully unsaturated. As such, the recitation of an alkylgroup includes both alkenyl and alkynyl groups. The alkyl can be amonovalent hydrocarbon radical, as described and exemplified above, orit can be a divalent hydrocarbon radical (i.e., alkylene).

The term “alkenyl” refers to a monovalent branched or unbranchedpartially unsaturated hydrocarbon chain (i.e. a carbon-carbon, sp²double bond). In some embodiments, an alkenyl group can have from 2 to 6carbon atoms, or 2 to 4 carbon atoms. Examples include, but are notlimited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and thelike. The alkenyl can be unsubstituted or substituted.

The term “alkynyl” refers to a monovalent branched or unbranchedhydrocarbon chain, having a point of complete unsaturation (i.e. acarbon-carbon, sp triple bond). In some embodiments, the alkynyl groupcan have from 2 to 6 carbon atoms, or 2 to 4 carbon atoms. This term isexemplified by groups such as ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, andthe like. The alkynyl can be unsubstituted or substituted.

The term “halo” refers to fluoro, chloro, bromo, and iodo. Similarly,the term “halogen” refers to fluorine, chlorine, bromine, and iodine.

As used herein, the term “substituted” is intended to indicate that oneor more (e.g., 1, 2, 3, 4, or 5; in some embodiments 1, 2, or 3; and inother embodiments 1 or 2) hydrogens on the group indicated in theexpression using “substituted” is replaced with a selection from theindicated group(s), or with a suitable group known to those of skill inthe art, provided that the indicated atom's normal valency is notexceeded and that the substitution results in a stable compound.Suitable indicated groups include, e.g., alkoxy, oxo, halo, haloalkyl,hydroxy, hydroxyalkyl, amino, alkylamino, dialkylamino, nitro, andcyano. Additionally, the suitable indicated groups can include, e.g.,—X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN,—N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —C(═O)R, —C(═O)X, —C(O)OR, where eachX is independently a halogen (“halo”): F, Cl, Br, or I; and each R isindependently H or alkyl. As would be readily understood by one skilledin the art, when a substituent is oxo (═O), or the like, then twohydrogen atoms on the substituted atom are replaced.

Synthesis

In some embodiments, the present invention provides a method forsynthesizing trehalose analogues comprising contacting a glucoseanalogue with a trehalose synthase, a magnesium salt, and amonosaccharide donor. In some embodiments, the present inventionprovides trehalose analogues quickly, e.g. in about 1 hour, in a highyield, e.g. up to 99%, in a single step from readily available glucoseanalogues.

In some embodiments, the monosaccharide donor is a nucleotidediphosphate monosaccharide, such as UDP-glucose, ADP-glucose,GDP-glucose, UDP-galactose, or GDP-mannose. For example, the nucleotidediphosphate may be uridine diphosphate (UDP), guanosine diphosphate(GDP), or adenosine diphosphate (ADP) and the monosaccharide may beglucose, galactose, allose, or mannose. In some embodiments, themonosaccharide may be labeled, e.g. with a radioactive or other isotope,such as ¹¹C, ¹³C, ¹⁴C, ²H, ³H, ¹⁵O, ¹⁸O, ¹³N, ¹⁵N, ³⁵S ¹⁸F and ¹²⁵I. Inother embodiments, the monosaccharide donor is glucosyl fluoride.

The magnesium salt is suitably magnesium chloride. The reaction mayfurther comprise a buffer, such as HEPES, Tris, or other commonbiological buffers. In some embodiments, the reaction is performed at apH of about 7 to about 7.5.

In some embodiments, the methods of the present invention may provide ayield of at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 97%, at least about 98%, or at leastabout 99%. In some embodiments, the methods of the present invention maybe performed in less than about 1 hour, less than about 2 hours, lessthan about 3 hours, less than about 6 hours, less than about 12 hours,or less than about 24 hours.

In some embodiments, the trehalose synthase is thermostable. In someembodiments, the trehalose synthase is unidirectional, meaning that itdoes not degrade trehalose. In some embodiments, the trehalose synthaseis from Thermoproteus tenax. Suitable trehalose synthases include, butare not limited to, those in Thermoccus litoralis, Pyrococcushorikoshii, Thermoproteus tenax, Rubrobacter xylanphilus, Pimelobactersp., Thermus aquaticus, Sulfolobus sp. The trehalose synthase may beisolated and purified from E. coli by techniques known to one ofordinary skill in the art.

In some embodiments, the glucose analogue is a monosaccharide. Theanalogue may be a natural sugar, such as mannose, galactose, or allose,or a non-natural sugar, such as a modified glucose, modified mannose,modified galactose, or a modified allose. In some embodiments, theglucose analogue is modified at the 2-, 3-, 4- and/or 6-positions. Insome embodiments, the glucose analogue may contain stereochemicalmodifications. Certain glucose analogues suitable for use in the presentinvention contain both substituent modifications and stereochemicalmodifications. In some embodiments, the glucose analogue contains one ormore of the following modifications: a stereochemical modification; ahalo substituent, such as fluoro or chloro; a deoxy modification; anazido substituent; a hydroxyl substituent; a ring sulfur; an isotope.

In some embodiments, the glucose analogue may contain one or moreisotopes, such as, but not limited to, ¹¹C, ¹³C, ¹⁴C, ²H, ³H, ¹⁵O, ¹⁸O,¹³N, ¹⁵N, ³⁵S, ¹⁸F and ¹²⁵I. In some embodiments, one or more of theisotopes may be a radioactive isotope. In some embodiments, an isotopemay be located at any position in the glucose analogue. For example, thering may contain an isotope. In some embodiments, an isotope may befound in one or more of R¹, R², R³ and R⁴. In some embodiments, anisotope may be located in the ring and in one or more of R¹, R², R³ andR⁴.

In some embodiments, the glucose analog may be further substituted by adetectable moiety that is detectable upon exposure to an externalstimulus. In some embodiments, the detectable moiety may be a reactivechemical handle (e.g. a biorthoganol tag such as azido, alkynyl, orcylcopropenyl), a fluorophore, a luminescent moiety (e.g. achemiluminescent moiety, a thermoluminescent moiety, or anelectroluminescent moiety), or a phosphorescent moiety. Suitablefluorophores include, but are not limited to, fluoresceins, xanthenes,cyanines, naphthalenes, coumarins, oxadiazoles, pyrenes, oxazines,acridines, arylmethines, Alexa Fluors and tetrapyrroles. In someembodiments, the detectable moiety is attached to the glucose analoguethrough a reactive chemical moiety such as azido, alkynyl orcyclopropenyl.

The glucose analogue may suitably be a compound of Formula (I):

wherein

-   -   X is O, S, Se, PH, P(O)H, or P(O)OH;    -   R¹, R² and R³ are independently selected from H, halo, —OR,        —NR₂, —NR₃+, —NHCOR, —SR, —SCOR, —OSO₃ ⁻, —OPO₃ ²⁻, —SeR,        —SeCOR, —N₃ ⁺, —CN, —NC, C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆        alkynyl;    -   R⁴ is OH, —NHR, —SH, or —SeH; and    -   each R is independently selected from H, C₁₋₆ alkyl, C₂₋₆        alkenyl or C₂₋₆ alkynyl.

The glucose analogue may suitably be a compound of Formula (II):

wherein

-   -   X is O, S, Se, PH, P(O)H, or P(O)OH;    -   R¹, R² and R³ are independently selected from H, halo, —OR,        —NR₂, —NR₃ ⁺, —NHCOR, —SR, —SCOR, —OSO₃ ⁻, —OPO₃ ²⁻, —SeR,        —SeCOR, —N₃, —CN, —NC, C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆        alkynyl;    -   R⁴ is OH, —NHR, —SH, or —SeH; and    -   each R is independently selected from H, C₁₋₆ alkyl, C₂₋₆        alkenyl or C₂₋₆ alkynyl.

As described herein, all glucose and trehalose analogues include allpossible isotopic substitutions. For example, H may be in any isotopicform, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form,including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including¹⁶O and ¹⁸O; and the like.

Exemplary substrates and their products are shown in FIGS. 1A-D.

Use of Trehalose Analogues

The present invention also provides a simple method for the synthesisand use of chemical probes to detect live mycobacteria, e.g.,Mycobacterium tuberculosis, and possibly other trehalose-containingorganisms/pathogens. In some embodiments, this method is performedwithout purification of the enzymatic reaction. This is referred to as“one-pot metabolic labeling.” One-pot metabolic labeling capitalizes onthe biocompatible nature of enzymatic reactions. After performing theenzymatic reaction as described above, the reaction mixture containingthe trehalose analogue product can be directly added to a samplecontaining live mycobacteria (or possibly other trehalose-containingorganisms/pathogens) with no or minimal processing or purification ofthe enzymatic reaction (as shown in Scheme 2). The mycobacteria (orpossibly other trehalose-containing organisms/pathogens) take up thetrehalose analogue from the aqueous enzymatic reaction mixture, leadingto labeling of the cell. If the trehalose analogue is detectable (e.g.,¹⁴C-, ¹⁸F-, ³H, or N₃-labeled or labeled with a reactive chemicalhandle, a fluorophore, a luminescent moiety or a phosphorescent moiety),then mycobacteria (or possibly other trehalose-containingorganisms/pathogens) are readily detected using traditional analyticalmethods known to one skilled in the art, such as positron emissiontopography (PET), autoradiographic analysis, scintillation detection,magnetic resonance imaging (MRI), nuclear magnetic resonance (NMR),x-ray photography, computed tomography (CT), single photon emissioncomputed tomography (SPECT) and fluorescence microscopy. In someembodiments, one-pot metabolic labeling provides trehalose analoguesynthesis, metabolic labeling, and imaging of mycobacteria in about 1hour, or about 2 hours or about 3 hours or about 5 hours or about 10hours. This one-pot approach is useful for rapidly synthesizing andadministering trehalose analogues to mycobacteria (or possibly othertrehalose-containing organisms/pathogens). See FIG. 4 .

The present invention also provides a method of determining the presenceof mycobacteria species in a subject or sample. In some embodiments, themethod comprises adding a labeled trehalose analogue to an organism orsample. The presence of mycobacteria is then determined by detecting thelabeled trehalose analogue. Without wishing to be bound by theory, thelabeled trehalose analogue accumulates in mycobacteria via atrehalose-specific transport protein, such as SugABC-LpqY. In someembodiments, the trehalose analogue is labeled with an isotope, such as¹⁸F. In some embodiments, the trehalose analog is labeled with adetectable moiety such as a fluorophore, a luminescent moiety or aphosphorescent moiety.

The methods of the present invention are useful in cells grown inculture medium, i.e., in vitro, or in cells within animals, e.g., livinganimals, i.e., in vivo. For research purposes, for measurements in cellsin vivo, a trehalose analogue as described herein is administered, e.g.,injected into the animal or added to an aqueous solution, e.g., water,or food consumed by the animal, to the animal. The methods of thepresent invention may be used in intact cells or in isolated organelles.

Cells may be mammalian cells, including but not limited to human,simian, murine, canine, bovine, equine, feline, ovine, caprine or swinecells, or prokaryotic cells, or cells from two or more differentorganisms, or cell lysates or supernatants thereof. In certain aspects,the cell may be in an animal, or physiological fluid, e.g., blood,plasma, urine, mucous secretions or the like.

In some embodiments, the sample is a biological sample such as tissue,cells, bodily fluid, sputum, cerebrospinal fluid, pericardial fluid,synovial fluid, ascitic fluid, blood, bone marrow, urine, or feces.

Trehalose Analogues

The present application also provides various trehalose analogues. Insome embodiments, the trehalose analogue is a compound according toFormula (III) or Formula (IV):

wherein

-   -   X is O, S, Se, PH, P(O)H, or P(O)OH;    -   R¹, R² and R³ are independently selected from H, halo, —OR,        —NR₂, —NR₃ ⁺, —NHCOR, —SR, —SCOR, —OSO₃ ⁻, —OPO₃ ²⁻, —SeR,        —SeCOR, —N₃, —CN, —NC, C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆        alkynyl;    -   R⁴ is OH, —NHR, —SH, or —SeH; and    -   each R is independently selected from H, C₁₋₆ alkyl, C₂₋₆        alkenyl or C₂₋₆ alkynyl; with the proviso that the analogue is        not trehalose.

Trehalose analogues contemplated by the present invention include, butare not limited to:

It is specifically understood that any numerical value recited herein(e.g., ranges) includes all values from the lower value to the uppervalue, i.e., all possible combinations of numerical values between thelowest value and the highest value enumerated are to be considered to beexpressly stated in this application. For example, if a concentrationrange is stated as 1% to 50%, it is intended that values such as 2% to40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in thisspecification. These are only examples of what is specifically intended.

EXAMPLES Example 1: Materials and Methods for Examples 2 and 3

General Experimental Section: Materials and reagents were obtained fromcommercial sources without further purification. Most glucose analogueswere obtained from Sigma-Aldrich (2-GlcAz, 2-DeoxyGlc, 6-GlcAz),CarboSynth (2-FluoroGlc, 3-DeoxyGlc, 3-FluoroGlc, 4-DeoxyGlc,4-FluoroGlc, 6-DeoxyGlc, 6-FluoroGlc), or Santa Cruz Biotechnology(5-ThioGlc). 3-GlcAz and 4-GlcAz were chemically synthesized accordingto literature-reported procedures. Chromatographic analysis of reactionswas performed using a Perkin Elmer Series 200 high-performance liquidchromatography (HPLC) system equipped with a Perkin Elmer Series 200refractive index detector. High-resolution electrospray ionization (HRESI) mass spectra were obtained in negative mode using a Waters LCTPremier XE using raffinose as the lock mass. ¹H NMR and ¹³C NMR spectrawere recorded on a Varian Inova 500 MHz NMR. Analytical TLC wasperformed on glass-backed silica gel 60 Å plates (thickness 250 m) fromDynamic Adsorbents and detected by charring with 5% H₂SO₄ in EtOH.Column chromatography was performed using flash-grade silica gel 32-63μm (230-400 mesh) from Dynamic Adsorbents.

Cloning, Expression, and Purification of TreT: The treT gene encodingtrehalose synthase from Thermoproteus tenax was codon-optimized for E.coli and synthesized by Life Technologies. The optimized treT sequencewas cloned into the pBAD/His A vector using restriction sites KpnI andSacI on the N-terminus and C-terminus, respectively, and verified bysequencing. The resultant pBAD-HisA TreT plasmid encodes an N-terminal6×histidine tag on the protein. The plasmid was transformed into Top10chemically competent E. coli and plated on LB agar containing 100 μg/mLampicillin.

To express the TreT protein, a 3 mL LB/ampicillin culture was inoculatedwith a single colony and grown overnight in a shaking incubator at 37°C. The following day, this 3 mL culture was used to inoculate a 600 mLculture of Terrific Broth with 100 μg/mL ampicillin. Once the inoculatedculture reached mid-log phase, it was induced with arabinose (finalconcentration of arabinose=1 mM) and grown in a shaking incubatorovernight at 37° C.

Cells were then spun at 4000×g and the pellets resuspended in 20 mL ofEquilibration/Lysis/Wash buffer (300 mM NaCl, 50 mM NaH₂PO₄, 20 mMimidazole). Resuspended pellets were sonicated using a Fisher ScientificSonifier (3×45 s, 75% amplitude). Sonicated cells were spun at 15,000×gfor 20 minutes to clarify the lysate. Next, lysates were loaded on a 5mL Bio-Rad Bio-scale Mini Profinity immobilized metal affinitychromatography (IMAC) cartridge at 5 mL/minute and washed until the A₂₈₀so reached background levels. TreT protein was eluted using a gradientcontaining high imidazole buffer (300 mM NaCl, 50 mM NaH₂PO₄, 250 mMimidazole). Protein samples were concentrated in an Amicon Ultra 15 mLconcentrator with a 10,000 molecular weight cutoff. Elution buffer wasexchanged for 300 mM NaCl, 50 mM HEPES, pH 7. Protein samples wereanalyzed by SDS-PAGE to verify protein purity and concentration wasassessed using UV-Vis spectroscopy.

General Method for Enzymatic Reactions: Concentrated (OX) stocksolutions of the reaction reagents in 50 mM HEPES buffer (pH 7.4)included glucose analogues (100 mM), UDP-Glc (400 mM), and MgCl₂ (200mM). Microscale reactions (50 μL) were performed by addition of 5 μL ofeach reagent and an appropriate volume of HEPES buffer, followed byaddition of TreT to a final concentration of 9.8 M to initiate thereaction. Reactions were incubated at 70° C. with gentle shaking for 1h, after which an equal volume of ice-cold HPLC-grade acetone (50 μL)was added to quench the reaction. After cooling at −20° C. for 1 h,reactions were centrifuged at 11,900 rpm for 20 min, and the supernatantwas collected and directly analyzed by HPLC (column: Imtakt UK-Amino250×46 mm at 50° C.; mobile phase: isocratic elution with pre-mixed 80%acetonitrile in water; flow rate: 0.4 mL/min; detection: refractiveindex). Yields were calculated by comparing relative peak areas ofsubstrates and products.

¹H NMR, ¹³C NMR, and HR ESI MS Data for Semi-Preparative ReactionProducts: Semi-preparative reactions were performed exactly as describedabove only in larger final volumes (1.5-2.0 mL). After incubation for 1h at 70° C., reactions were quenched by addition of an equal volume ofcold acetone, cooled at −20° C. for 1 h, and centrifuged at 11,900 rpmfor 20 min. The supernatant was collected and concentrated in vacuum,and the resulting residue was resuspended in CH₂Cl₂/CH₃OH (1:1) andpassed through a silica plug using CH₂Cl₂/CH₃OH (1:1) as the eluent. Ifnecessary, further purification was accomplished by silica gel columnchromatography using an elution solvent of CH₂Cl₂/CH₃OH (2:1 or 2.5:1).After concentration, the purified products were redissolved in water,filtered (0.2 μm), and concentrated to give white solids.

2-Deoxy-2-fluoro-α,α-D-trehalose (2-FluoroTre): From 3.6 mg 2-FluoroGlc,obtained 6.6 mg 2-FluoroTre (97%). ¹H NMR (500 MHz, D₂O): δ 5.41 (d,J=3.5 Hz, 1H), 5.19 (d, J=3.5 Hz, 1H), 4.48 (ddd, J_(H,H)=3.5, 9.5 Hz,J_(H,F)=49 Hz, 1H), 4.10 (dt, J_(H,H)=9.5 Hz, J_(H,F)=13.5 Hz, 1H),3.88-3.72 (m, 7H), 3.63 (dd, J=3.5, 10 Hz, 1H), 3.49 (t, J=9.0 Hz, 1 H),3.43 (t, J=10 Hz, 1H). ¹³C NMR (125 MHz, D₂O): δ 93.97, 91.15 (d,J_(C,F)=22 Hz), 89.44 (d, J_(C,F)=188 Hz), 72.54, 72.16, 71.05 (d,J_(C,F)=17 Hz), 70.89, 69.51, 69.02, 68.96, 60.43, 60.22. HR ESI MSnegative mode: calcd. for C₁₂H₂₁ClFO₁₀ [M+Cl]⁻ m/z, 379.0807; found,379.0800.

2-Deoxy-α,α-D-trehalose (2-DeoxyTre): From 3.3 mg of 2-DeoxyGlc,obtained 6.2 mg 2-DeoxyTre (94%). ¹H NMR (500 MHz, D₂O): δ 5.28 (d,J=3.0 Hz, 1H), 5.13 (d, J=3.5 Hz, 1H), 4.03 (m, 1H), 3.86-3.72 (m, 6H),3.67 (m, 1H), 3.59 (dd, J=3.5, 9.5 Hz, 1H), 3.42 (t, J=9.0 Hz, 1H), 3.38(t, J=9.0 Hz, 1H), 2.20 (dd, J=5.0, 13 Hz, 1H), 1.75 (dt, J=3.5, 13 Hz).¹³C NMR (125 MHz, D₂O): δ 93.19, 92.30, 72.64, 72.61, 72.12, 70.90,69.59, 67.88, 60.57, 60.43, 36.27. HR ESI MS negative mode: calcd. forC₁₂H₂₂ClO₁₀ [M+Cl]⁻ m/z, 361.0902; found, 361.0884.

3-Deoxy-3-fluoro-α,α-D-trehalose (3-FluoroTre): From 3.6 mg of3-FluoroGlc, obtained 6.5 mg 3-FluoroTre (96%). ¹H NMR (500 MHz, D₂O): δ5.23 (t, J=3.5 Hz, 1H), 5.17 (d, J=4.0 Hz, 1H), 4.74 (dt, J_(H,H)=9.5Hz, J_(H,F)=55 Hz, 1H), 3.92 (m, 1H), 3.87-3.73 (m, 7H), 3.63 (dd,J=3.5, 9.5 Hz, 1H), 3.43 (t, J=9.5 Hz, 1H). ¹³C NMR (125 MHz, D₂O): δ94.27 (d, J_(C,F)=178 Hz), 93.43 (d, J_(C,F)=10.5 Hz), 93.35, 72.41,72.14, 71.59 (d, J_(C,F)=6.6 Hz), 70.88, 69.53, 69.45 (d, J_(C,F)=20Hz), 67.80 (d, J_(C,F)=17 Hz), 60.40, 60.00. HR ESI MS negative mode:calcd. for C₁₂H₂₁ClFO₁₀[M+Cl]⁻ m/z, 379.0807; found, 379.0794.

6-Azido-6-Deoxy-α,α-D-trehalose (6-TreAz): From 4.1 mg of 6-GlcAz,obtained 6.7 mg 6-TreAz (92%). ¹H NMR (500 MHz, D₂O): δ 5.05 (d, J=4.5Hz, 1H), 5.04 (d, J=4.0 Hz, 1H), 3.82 (ddd, J=2.5, 6.0, 10.5 Hz, 1H),3.72-3.67 (m, 4H), 3.61 (dd, J=5.0, 12 Hz, 1H), 3.55-3.49 (m, 3H), 3.42(dd, J=5.5, 13.5 Hz, 1H), 3.31 (t, J=9.5 Hz, 1H), 3.30 (t, J=9.0 Hz,1H). ¹³C NMR (125 MHz, D₂O): δ 93.53, 93.33, 72.43, 72.23, 72.12, 70.91,70.89, 70.86, 70.38, 69.57, 60.40, 50.78. HR ESI MS negative mode:calcd. for C₁₂H₂₁ClN₃O₁₀ [M+Cl]⁻ m/z, 402.0915; found, 402.0922.

5-Deoxy-5-Thio-α,α-D-trehalose (5-ThioTre): From 3.9 mg of 5-ThioGlc,obtained 6.5 mg 5-ThioTre (92%). ¹H NMR (500 MHz, D₂O): δ 5.41 (d, J=4.0Hz, 1H), 4.98 (d, J=3.5 Hz, 1H), 3.93 (dd, J=3.0, 10 Hz, 1H), 3.89-3.77(m, 6H), 3.75 (dd, J=5.0, 11 Hz, 1H), 3.67-3.63 (m, 2H), 3.44 (t, J=9.5Hz, 1H), 3.13 (m, 1H). ¹³C NMR (125 MHz, D₂O): δ 93.48, 76.99, 74.76,73.62, 73.53, 72.71, 72.24, 70.92, 69.63, 60.43, 59.90, 42.87. HR ESI MSnegative mode: calcd. for C₁₂H₂₂ClO₁₀S [M+Cl]⁻ m/z, 393.0622; found,393.0606.

One-Pot Metabolic Labeling and Imaging of M. smegmatis: Cultures of M.smegmatis wild type, ΔsugC mutant, or ΔsugC::sugC complement weregenerated by inoculating a single colony from a freshly streaked agarplate (with appropriate antibiotic, if needed) into 3 mL Middlebrook 7H9liquid medium supplemented with ADC (albumin, dextrose, and catalase),0.5% glycerol, and 0.05% Tween-80 in a culture tube. Starter cultureswere incubated at 37° C. with shaking until reaching log phase.Meanwhile, using the procedure described above, a 50 μL TreT reactionemploying 6-GlcAz (10 mM) as the substrate was carried out to synthesize6-TreAz (+TreT sample). A control reaction lacking TreT was run inparallel (−TreT control). After incubation at 70° C. for 1 h, thereactions were stopped by placing on ice. Given the observedquantitative conversion for this reaction (FIG. 1B, Entry 16), a 6-TreAzconcentration of 10 mM was assumed in the +TreT sample. For the −TreTcontrol, a 6-GcAz concentration of 10 mM was assumed. The aqueousreaction mixtures were directly diluted into M. smegmatis wild type,ΔsugC mutant, or ΔsugC::sugC complement growing in 7H9 liquid medium toa final azido sugar concentration of 25 μM and a culture density ofOD₆₀₀=0.15. Control experiments in which bacterial strains were culturedonly in 7H9 liquid medium were run in parallel. Bacteria were incubatedat 37° C. for 4 h (final densities of all cultures wereOD₆₀₀=0.60-0.64), after which cells were pelleted (5000 rpm for 5 min)and washed three times with phosphate-buffered saline supplemented with0.5% BSA (PBS-B), then fixed by treatment with 4% paraformaldehyde inPBS for 15 min. Fixed cells were pelleted and washed once with PBS-B,then reacted with alkyne-modified carboxyrhodamine 110 fluorophore(Click Chemistry Tools) via Cu-catalyzed azide-alkyne cycloaddition(CuAAC) according to Breidenbach et al., Proc. Natl. Acad. Sci. U.S.A.(2010) 108, 3141 and Swarts et al., J. Am. Chem. Soc. (2012) 134, 16123,the entire contents of both of which are incorporated herein byreference. Following CuAAC, cells were pelleted and washed three timeswith PBS-B. 10 uL of bacteria resuspended in PBS were spotted onto amicroscope slide, lightly spread into a thin layer using the edge of acoverslip, and allowed to air dry in the dark. Fluoromount-G mountingmedium (SouthemBiotech) was applied, then cover slips were placed overthe sample and immobilized with adhesive. Microscopy was carried outusing an EVOS FL (Life Technologies) inverted microscope equipped with a100×1.4 numerical aperture Plan-Apochromat oil immersion lens.Fluorescence imaging was performed using a GFP LED light cube (maximumexcitation/emission=470/510 nm). Images were captured with a Sony ICX445CCD camera and processed using the FIJI distribution of ImageJ. Imageacquisition and processing were performed identically for all samples.

Genetic Complementation of the M. smegmatis ΔsugC Mutant Strain: ThesugC gene (MSMEG_5058) was amplified from genomic DNA of M. smegmatismc²155 by PCR using the oligonucleotide pair5′-TTTTTTTAATTAAATGGCCGAAATTGTGTTGGATCG-3′ (SEQ ID NO:1) and5′-TTTTTAAGCTTCACCCGGCGTCCGGCCCCGCCG-3′ (SEQ ID NO:2) and cloned usingthe restriction enzymes PacI and HindIII (underlined) into thesingle-copy integrative plasmid pMV361(Apra)-PacI, a derivative ofpMV361(Kan) containing an apramycin resistance gene and a unique PacIrestriction site. The resulting plasmid pMV361(Apra)::MSMEG_5058 wasthen transformed by electroporation into the M. smegmatis ΔsugC mutantyielding the complemented mutant strain M. smegmatis ΔsugC::sugC withstable chromosomal integration of the plasmid into the mycobacteriophageL5 attB site, providing constitutive sugC gene expression from the HSP60promoter. Solid medium containing 50 mg/L hygromycin and 10 mg/Lapramycin was used for selection.

Example 2: Trehalose Analogue Synthesis

Studies were performed to synthesize trehalose analogues using trehalosesynthase TreT from the hyperthermophile Thermoproteus tenax, forexample, as shown in Scheme 1 below. Specifically, scheme 1 shows thesynthesis of trehalose from Glc and UDP-Glc by TreT from T. tenax.

TreT was expressed and purified from E. coli and screened for reactivityusing a panel of monofunctionalized Glc analogues (FIGS. 1A, 1 i, and1C). The Glc analogues that were tested contained fluoro-, deoxy-,azido-, and stereochemical modifications occurring at all positions ofthe sugar ring, which afforded a systematic evaluation of TreT substratespecificity. Reactions were performed in 50 mM HEPES buffer (pH 7.4)containing 10 mM Glc analogue, 40 mM UDP-Glc, 20 mM MgCl₂, and 9.8 MTreT (reaction volume 50 μL). The reactions were incubated at 70° C.with gentle shaking for 1 h, quenched by addition of cold acetone, andanalyzed by HPLC and high-resolution ESI mass spectrometry as describedabove in Example 1.

The Glc analogues which were evaluated were well-tolerated by TreT(FIGS. 1A, 1B, and 1C). In most cases, the corresponding trehaloseanalogue products were generated in excellent yield after only 1 h,which highlighted the efficiency, rapidity, and generality of themethod. Fluoro-, deoxy-, azido-, and stereochemical modifications of theGlc 2-, 3-, and 6-positions were generally accepted, except for azidosubstitution at the 2-position and inversion of the 3-OH group. Glcanalogues bearing 4-position alterations were poor substrates (N.D.-26%yield), indicating a strict specificity at this position. Finally,5-thio-D-glucose, the sole 5-position-modified Glc analogue that wastested, was converted to 5-thio-trehalose in quantitative yield.

Selected reactions were run on a semi-preparative scale (5-10 mg) toevaluate the scalability of the method and to confirm product structureby NMR spectroscopy. Semi-preparative reactions were performed asdescribed above and the products were readily purified by silica gelchromatography. Consistent with the small-scale results, 2-FluoroTre,2-DeoxyTre, 3-FluoroTre, 6-TreAz, and 5-ThioTre were obtained inisolated yields of 92-97%. ¹H and ¹³C NMR analysis established theproduct structures, including the assignment of 1,1-α,α-stereochemistryfor newly formed glycosidic bonds (see Example 1).

Example 3: Single-Day Probe Synthesis, Metabolic Labeling, and Imaging

To evaluate the applicability of the method described in Example 2 toimaging mycobacteria, a one-day experiment was performed thatencompassed probe synthesis, metabolic labeling, and imaging of M.smegmatis, which is an avirulent model organism frequently used intuberculosis (TB) research (FIG. 2A).

First, TreT was used to convert commercially available6-azido-6-deoxy-D-glucose (6-GlcAz) to 6-TreAz (FIG. 1B, Entry 16), anestablished chemical reporter for metabolic labeling of mycobacterialglycolipids. Next, the biocompatibility of enzymatic reactions wasutilized by directly diluting the aqueous TreT reaction mixture intolive M. smegmatis cell suspension, which is referred to herein as“one-pot metabolic labeling.” After incubation for 4 h, 6-TreAz-labeledmycobacteria were washed, fixed, and reacted with an azide-reactivefluorophore, alkyne-488, via Cu-catalyzed azide-alkyne cycloaddition(CuAAC).

As shown in FIG. 2B, fluorescence microscopy revealed strong labeling ofwild-type M. smegmatis that was treated with a reaction mixturecontaining 6-TreAz (+TreT). No fluorescence was observed when bacteriawere treated with a reaction mixture lacking TreT (−TreT), whichconsisted only of unreacted substrates.

The same experiments were performed in M. smegmatis ΔsugC, a mutantmissing the trehalose transporter required for 6-TreAz uptake andlabeling, as well as its complement, M. smegmatis ΔsugC::sugC.Fluorescence was abolished in the ΔsugC mutant and restored in thecomplement, confirming that 6-TreAz labeling proceeded via the trehalosetransporter pathway.

Additionally, direct treatment of M. smegmatis with TreT reactionmixture had no effect on bacterial growth or appearance. Thisoperationally simple experiment provided a model for rapidly preparingand administering trehalose-based chemical probes. By contrast,traditional methods for synthesizing and purifying these compounds canrequire several weeks of work by a trained chemist in a well-equippedsynthesis laboratory.

Example 4: Uptake of Fluoro Trehalose by the Trehalose Transporter inMycobacteria

Cultures of M. smegmatis wild type, ΔsugC mutant, or ΔsugC::sugCcomplement were generated by inoculating a single colony from a freshlystreaked agar plate (with appropriate antibiotic, if needed) into 3 mLMiddlebrook 7H9 liquid medium supplemented with ADC (albumin, dextrose,and catalase), 0.5% glycerol, and 0.05% Tween-80 in a culture tube.Starter cultures were incubated at 37° C. with shaking until reachinglog phase. Cells were then cultured at 37° C. in the presence or absenceof 2-, 3-, 4-, or 6-fluoro trehalose (100 μM) in 7H9 liquid medium for 1h, then centrifuged and washed (5000 rpm for 5 min) three times inultrapure water. The washed cells were resuspended in ultrapure waterand boiled for 4 h, then centrifuged. The supernatant was collected anddried on a speedvac to yield water-soluble cytosolic metabolites (e.g.,trehalose and fluoro trehaloses, if present), which were subsequentlytrimethylsilyl (TMS)-derivatized using N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) in pyridine and analyzed by gaschromatography-mass spectrometry (GC-MS).

FIGS. 3A-3D show gas chromatograms for uptake of purified 2-, 3-, 4-,and 6-fluoro trehalose. The corresponding fluoro trehalose derivativewas observed in cytosolic extracts from wild-type M. smegmatis treatedwith 2-, 3-, and 6-fluoro trehalose, but not 4-fluoro trehalose. Nouptake was observed for any analogues in the M. smegmatis ΔsugC mutant,while uptake was restored for each analogue in the ΔsugC::sugCcomplement. These data indicate that uptake of the fluoro trehaloseanalogues by mycobacteria is dependent on the trehalose transporterSugABC-LpqY. FIG. 3E shows gas chromatograms in a similar experiment,but in this case the 2-fluoro trehalose synthesized and administeredusing the rapid “one-pot metabolic labeling” method described above.Therefore, fluoro trehaloses can be rapidly synthesized by TreT in 60minutes and immediately administered to mycobacteria, where they canselectively accumulate in the cytosol via the trehalose transporter.

In summary, Examples 1-4 demonstrated that the thermostable trehalosesynthase TreT from T. tenax converted a broad variety of Glc analoguesinto trehalose analogues in a single step in high yield (up to >99%) in1 h. The TreT reaction was biocompatible and rapid (i.e., 1 h), andthus, allowed for administration of trehalose-based chemical probes tomycobacteria and imaging of mycobacteria with no effect on bacterialgrowth or appearance. Accordingly, probe synthesis, metabolic labeling,and imaging of the mycobacteria was accomplished in a single day.

Various features and advantages of the invention are set forth in thefollowing claims.

What is claimed is:
 1. A method for synthesizing a trehalose analoguecomprising: contacting a glucose analogue of Formula (I) with amonosaccharide donor, a magnesium salt, and a trehalose synthase fromThermoproteus tenax,

wherein X is O; R¹, R² and R³ are independently selected from H, fluoro,—OR, and —N₃; R⁴ is OH; and each R is independently selected from H andC₁₋₆ alkyl; with the proviso that either the glucose analogue is notglucose or the monosaccharide donor is not a glucose donor.
 2. Themethod of claim 1, wherein the glucose analogue is not glucose, themonosaccharide donor is a glucose donor, and the trehalose analogue is acompound of formula (III)


3. The method of claim 2, wherein R is H.
 4. The method of claim 2,wherein the trehalose analogue is


5. The method of claim 4, wherein the trehalose analogue is


6. The method of claim 4, wherein the trehalose analogue is


7. The method of claim 4, wherein the trehalose analogue is


8. The method of claim 5, wherein the trehalose analogue is labeled with¹⁸F.
 9. The method of claim 6, wherein the trehalose analogue is labeledwith ¹⁸F.
 10. The method of claim 7, wherein the trehalose analogue islabeled with ¹⁸F.
 11. The method of claim 8, wherein the glucose donoris selected from the group consisting of a nucleotide diphosphateglucose and glucosyl fluoride.
 12. The method of claim 11, wherein theglucose donor is the nucleotide diphosphate glucose.
 13. A method ofpreparing a trehalose analogue of formula

comprising: reacting a compound of formula

with UDP-glucose, a magnesium salt, and a trehalose synthase fromThermoproteus tenax, wherein each R is independently OH, F, H, or N₃; Xis O; and

is not glucose.
 14. The method of claim 13, wherein:

and the trehalose analogue is


15. The method of claim 13, wherein:

and the trehalose analogue is


16. The method of claim 13, wherein:

and the trehalose analogue is


17. The method of claim 13, wherein:

and the trehalose analogue is


18. The method of claim 14, wherein the trehalose analogue is labeledwith ¹⁸F.
 19. The method of claim 15, wherein the trehalose analogue islabeled with ¹⁸F.
 20. The method of claim 16, wherein the trehaloseanalogue is labeled with ¹⁸F.
 21. The method of claim 17, wherein thetrehalose analogue is labeled with ¹⁸F.